Our goal is to define the mechanisms of internalization and sorting in the early endocytic pathway. These mechanisms relate to important physiological processes, such as glucose transport and growth-factor receptor down regulation, which are disrupted in diseases such as diabetes and cancer, respectively. Proteins that interact with phosphatidylinositol 3-phosphate (PI3P) via their FYVE domain play an important yet incompletely understood role in endocytic trafficking. During the previous funding period we achieved a detailed understanding of the structural and cellular basis for the association of EEA1 to the surface of early endosomes through the interaction of its FYVE domain to PI3P, but many questions remain unanswered regarding the precise function of PI3P, EEA1 and other FYVE-domain containing proteins in endocytic trafficking. We have found that depletion of EEA1 profoundly inhibits EGF receptor (EGFR) degradation, yet has a negligible effect on transferrin internalization and recycling. These new results place EEA1 at a point in the endocytic pathway related to the trafficking of cargo such as the EGFR towards the multivesicular body (MVB) /lysosomal pathway, and suggest that other FYVE-domain containing proteins, such as Rabenosyn-5, may be more relevant for recycling of proteins such as the transferrin receptor and GLUT4 to the plasma membrane. Moreover, we have achieved the direct visualization of fluorescent ligand binding and internalization in live cells in real time, as well as the activation of GTPase Rab5 and the recruitment of EEA1 to early endosomes in response to EGF. In collaboration with Projects 1,3 and 4, as well as with the Imaging Core, we will use a combination of structural, biochemical and imaging technologies to test the following hypotheses: 1: That high and low affinity binding of EGF is due to the dynamic distribution of EGFRs among distinct plasma membrane sub domains. 2: That activated, ubiquitinated EGFR activates Rab5 through its interaction with Vps9 domain-containing proteins, and by mechanisms that include release of autoinhibition of RabexS. 3: That EEA1 interacting with activated RabS is required for EGFR degradation via the MVB-lysosomal pathway. 4: That sorting of the TfR and GLUT4 from EGFR depends on the relative activities of Rabs 4 and 5, and their mutually exclusive interactions with the dual PI3P-Rab binding proteins EEA1, RabenosynSand Rabip4.